In the female reproductive system, the menstrual cycle is a recurring cycle of physiological changes and is necessary for reproduction that occurs in reproductive-age females. Menstrual cycles are counted from the first day of menstrual flow, because the onset of menstruation corresponds closely with the hormonal cycle and because menstrual bleeding is a readily apparent event for the individual concerned. The menstrual cycle may be divided into several phases, and the length of each phase varies from woman to woman and from cycle to cycle. The phases as well as their average values are typically as follows: the menstrual phase, days 1-4; the follicular phase (also known as proliferative phase), days 5-13; ovulation, day 14; the luteal phase, days 15-26 and the ischemic phase, days 27-28.
The follicular phase is the phase of the menstrual cycle during which follicles in the ovary mature. Through the influence of a rise in follicle stimulating hormone (FSH), about five to seven tertiary-stage ovarian follicles are recruited for entry into the next menstrual cycle. As they mature, the follicles secrete increasing amounts of estradiol. When the egg has matured, it secretes enough estradiol to trigger the acute release of luteinizing hormone (LH). In the average cycle this “LH surge” starts around cycle day 12 and may last about 48 hours. The release of LH matures the egg and weakens the wall of the follicle in the ovary. This process leads to ovulation. Estrogen levels continue to increase and are at their highest just before the LH surge begins. Most sources agree that ovulation normally occurs 24-48 hours after the beginning of the LH surge, typically about 36 hours after the LH surge.
The luteal phase is the latter phase of the menstrual cycle and begins with the formation of the corpus luteum and ends in either pregnancy or luteolysis. The main hormone controlling this stage is progesterone, which is significantly higher during the luteal phase than other phases of the cycle. The length of the follicular phase, and consequently the length of the menstrual cycle may vary widely; some women have a follicular phase of 10 days, others 16 days, while the average is 14 days. The luteal phase, however, almost always takes the same number of days for each woman. Normal sperm life inside a woman ranges from 1-5 days. The most fertile period (the time during which sexual intercourse is most likely to result in pregnancy) covers the time from some 5 days before ovulation until 1-2 days after ovulation. In an average 28 day cycle with a 14-day luteal phase, this corresponds to the second and the beginning of the third week of the cycle.
Following conception, β-hCG is secreted by the placenta and signals the corpus luteum to continue progesterone secretion, thereby maintaining the thick lining (endometrium) of the uterus. β-hCG continues to be secreted until placenta is able to secrete its own progesterone. The hCG hormone level in the human body doubles approximately every 2.2 days during the first trimester of pregnancy. If the egg is not fertilized, and therefore no β-hCG is produced, the corpus luteum stops secreting progesterone and decays. Without progesterone the uterine lining is expelled through the vagina (menses) and the menstrual cycle begins once more. Detectable levels of hCG in urine start at 5 mIU/ml during the first week of gestation and rise to 100,000 mIU/ml at 2 to 3 months. Values decline to 10% to 15% of peak concentrations during 2nd and 3rd trimesters. Thus, there is a very extended range of analyte concentration values.
Simple assay devices for determining the levels of hCG in urine are widely available for over the counter and professional use. Such devices are used to determine whether or not a woman is pregnant by measurement of the presence or absence of the hormone β-hCG. Nowadays, women will typically purchase such a device if they suspect they may be pregnant before consulting a healthcare professional.
However, once a woman establishes or suspects that she is pregnant, it is useful for her to know how many weeks pregnant she is. This can help her and the doctor or midwife plan for the future in estimating a date of birth of the baby as well, as well as plan a diary for other key dates such as the twelve week scan. Typically the doctor or midwife will determine the extent of pregnancy based upon knowledge obtained from the woman of the first day of her last menstrual period based on a standard 28 day cycle. Whilst the luteal phase is known to be fairly constant at around 15 days, the follicular phase may vary widely from for example between 9-28 days. Cycle lengths may vary widely from one woman to another. Thus basing the extent of pregnancy based upon the first day of her last period may vary widely in accuracy. Furthermore this assumes that the woman has given the correct date for her last menstrual period. Consequently estimates by the doctor concerning the extent of pregnancy based solely upon the levels of hCG can be quite inaccurate. Whilst a typical hCG assay test assay test can provide an indication of whether the subject is either pregnant or not pregnant, it cannot itself provide a further indication of the extent of pregnancy without requiring further specific information such as the date of a last missed period.
Simple immunoassay devices for measuring levels of analytes such as hCG in urine are widely available, for example sold under the name of Unipath Clearblue® Lateral flow immunoassay devices are also described in EP291194. Whilst such devices are able to indicate the presence or absence of hCG above or below a certain threshold, typically 10 or 25 mIU, and indicate that a subject is either pregnant or not pregnant, they are unable for determining levels of analyte over an extended analyte range such as levels of hCG ranging from 10 to 250,000 mIU.
In principle it is possible to measure levels of hCG over an extended range using a simple lateral flow type device. However, merely an indication of the amount of hCG in concentration based units would be of limited use to a healthcare professional in the absence of information as to the date of the last missed period and almost certainly of no use to the lay user.
Lateral flow devices such as described by EP291194 have been developed and commercialised for detection of a number of analytes in fluid samples. Such devices typically comprise a porous carrier comprising a dried mobilisable labelled binding reagent capable of binding to the analyte in question, and an immobilised binding reagent also capable of binding to the analyte provided at a detection zone downstream from the labelled binding reagent. Detection of the immobilised labelled binding at the detection zone provides an indication of the presence of analyte in the sample.
Alternatively, when the analyte of interest is a hapten, the immunoassay device may employ a competition reaction wherein a labelled analyte or analyte analogue competes with analyte present in the sample for an immobilised binding reagent at a detection zone. Alternatively the assay device may employ an inhibition reaction whereby an immobilised analyte or analyte analogue is provided at a detection zone, the assay device comprising a mobilisable labelled binding reagent for the analyte.
A sandwich immunoassay is often the assay of choice when detecting analytes. However, a sandwich assay is not always possible, for example in the case of small molecules such as haptens which may not be large enough to allow the simultaneous binding thereto of two different binding partners. A dose-response curve prepared using a typical lateral flow device employing a sandwich immunoassay shows increasing levels of signal with increasing analyte up to the point where at higher analyte levels the curve tends to plateau. At yet higher analyte levels, the signal begins to decrease due to preferential capture at the detection zone of analyte which has not yet bound to labelled reagent. This phenomenon is known as the hook effect. Thus, especially if a quantitative or semi-quantitative assay result is required, sandwich immunoassays exhibit a limited assay range due to the fact that the signal amount or intensity observed at higher analyte levels may be the same, or even less, than that observed at lower analyte levels.
A competition or inhibition assay typically provides a high signal at zero or low levels of analyte. At increasing levels of analyte the signal level may still be high depending upon the amount of labelled binding species present compared to the amount of analyte. At still increasing levels of analyte, the signal starts to decrease as unbound analyte either competes with labelled analyte or analyte analogue for the immobilised binding reagent or binds to labelled binding reagent, lowering binding of the labelled binding reagent at the detection zone.
So, use of sandwich assays to measure analyte over an extended range incurs problems with respect to the hook effect. High analyte concentrations start producing a reduction in assay signal. Competition or inhibition assays result in the depletion in assay signal at high analyte concentrations and thus offer a limited range over which analyte can be measured.
Thus the above assay methods are not suitable for measuring levels of analyte over an extended analyte range.
A number of assay devices have been proposed to measure analytes over an extended range.
US2004/0197820 discloses a flow through porous carrier assay device for reducing the hook effect comprising a detection zone wherein the device may include a downstream calibration zone.
US2006/0019404 discloses an assay device with an extended dynamic range comprising a lateral flow test-strip comprising a plurality of detection zones with a progressively decreased sensitivity to analyte concentration. The assay device may comprise two carriers each having a plurality of detection zones. The amount of label/signal present at the plurality of detection zones is detected to determine the analyte concentration.
EP462376 discloses an assay device comprising a capture site and a conjugate recovery site wherein the conjugate recovery site receives and binds said conjugate or conjugate complexes which migrate through said capture site and wherein immobilised conjugate at both the conjugate recovery site and capture site is detected to determine the amount of the analyte of interest.
Lateral flow type assay devices employing an optically detectable labelled binding reagent for the analyte or analyte analogue are the assay device of choice for simple, disposable assays. However a large number of other assay devices and methods may be employed to detect analytes. For example, the assay method may comprise a binding reagent for the analyte labelled with an enzyme, radioactive, electrochemically active or magnetic label. The analyte may be detected by means of an enzyme and an electrochemical mediator. Simple devices for the detection of glucose comprising the enzyme glucose oxidase and an electron mediator such as ferro/ferricyanide are widely commercially available. Acoustic biosensor type devices and methods, for example measuring the resonant frequency of a quartz crystal following a binding event, allow for the determination of an analyte without need to provide a labelled binding reagent.